The positioning of Raf-1 as an essential cytoplasmic signal transducer downstream of ras and membrane-associated tyrosine kinases was initially suggested by data obtained with ras revertant cell lines, microinjection of inhibitory ras antibodies, and more recently, established by use of antisense and dominant negative raf mutants. Further, support for such a position of Raf-I in signal transduction comes from work demonstrating phosphorylation of Raf-I protein, resulting in a mobility shift and increased kinase activity in cells stimulated with a variety of cytokines, as well as after transformation with oncogenic versions of growth factors, growth factor receptors, and tyrosine kinase oncogenes. We have further examined the role of ras in the coupling of Raf-I to receptors with intrinsic tyrosine kinase activity by use of a dominant negative mutant of ras. Experiments with a dexamethasone-inducible dominant negative Ha-ras-Asnl7 mutant demonstrate that (1) transfection of NIH3T3 cells with the Ha-ras-Asnl7 mutant results in the inhibition of serum-induced proliferation, (2) serum induction of the Raf-I shift is blocked in these cells, and (3) this block in the Raf-I shift correlates with a block in the induction of Raf-I kinase activity. Two further avenues were followed for characterization of a Ras/Raf coupling event. Raf-I phosphorylation and kinase stimulation by Ras was examined under serum-free and low serum conditions. Activated Ras was found to depend on membrane signals for Raf-I activation. Furthermore, a potential synergistic interaction was tested between ras and raf by cotransfection of NIH3T3 cells and assay for transforming activity. Wildtype Raf-I alone is negative in this assay but greatly facilitates the ability of wild-type or activated Ras to induce transformation. We conclude that Ras controls the signal flow from activated receptors to Raf-I and depends on Raf-I for the transformation.